The chemical polycondensation of mononucleotides is an important method for preparing deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
Oligonucleotides are used to an increasing extent as inhibitors of gene expression (J. F. Milligan, M. D. Matteucci and J. C. Martin, J. Med Chem. 36(1993): 1923; E. Uhlmann and A. Peyman, Chemical Reviews 90: (1990) 543) or as ribozymes (e.g., D. Castanotto, J. J. Rossi, J. O. Deshler, Critical Rev. Eukar. Gene Expr. 2 (1992): 331), or in diagnosis as DNA probes (e.g., Beck und Koester, Anal. Chem. 62 (1990): 2258). There is therefore a great need for suitable methods for synthesizing such compounds.
The state of the art with regard to oligonucleotide synthesis is reviewed in E. Sonveaux, Bioorg. Chem. 14 (1986): 274; E. Uhlmann and A. Peyman, Chemical Reviews 90: (1990) 543, Beaucage and Iyer, Tetrahedron 49: (1993) 10441-10488.
A basic problem in the chemical synthesis of DNA or RNA is that of finding suitable protecting groups for the amino and hydroxyl groups of the nucleoside bases and the sugar residues. On the one hand, these protecting groups have to be stable under the conditions of the polycondensation reaction, i.e., during the reaction, and, on the other hand, they have to be sufficiently labile to enable them to be removed again at the end of the reaction without recleaving the phosphodiester bond (H. G. Khorana, Pure Appl. Chem. 17 (1968): 349).
Current practice in DNA synthesis essentially provides for three steps: (a) sequential synthesis of the variously protected nucleotides on a solid support; (b) cleavage of the synthesized oligonucleotides from the support; (c) deprotection of the oligonucleotides. While the synthesis of the oligonucleotide on the solid support takes place very rapidly--approximately one hour is required for a 20 mer--and cleavage from the support is also complete within an hour, the final deprotection of the oligonucleotide remains a problem. Standard oligonucleotide synthesis (e.g., M. Reddy, N. B. Hanna, F. Farooqui, WO 95/24413) provides for a treatment of approx. 6 h at 55.degree. C. with conc. NH.sub.3 when the conventional benzoyl, for dA and dC, and butyroyl for dG, protecting groups are employed. A whole series of protecting groups which are more sensitive to ammoniacal aminolysis than are the conventionally protected nucleotide derivatives have recently been proposed for speeding up this latter step (M. Reddy et al., see above; Beaucage and Iyer, see above). These protecting groups comprise, for example, the phenoxyacetal group (Schulhof et al., Nucl. Acids Res. 15 (1987): 397); the dimethylformamidine group (Vu et al., Tetrahedron Lett. 31 (1990): 7269), or the tert-butylphenoxyacetyl group (Sinha et al., Biochimie 75 (1993): 13), or phenylacetyl protecting groups as described in Reddy et al. (see above).
While the deprotecting time at 55.degree. C. is reduced to 15-60 minutes when these ammonia-labile protecting groups are employed, their use also suffers from disadvantages. In the first place, the lability of these groups also leads to instability toward the DNA synthesis conditions, for example during the capping step (Beaucage and Iyer, see above). Phenoxyacetyl protecting groups, for example, reduce the solubility of the nucleotide derivatives so that solvent mixtures have to be employed.
An additional criterion for the use of protecting groups for the exocyclic amino functions of the nucleobases is the purity of the resulting products. In the case of deprotecting procedures which use ammonia, and which are carried out after or during cleavage from the support, a mixture of oligonucleotide and eliminated protecting groups is always obtained. The oligonucleotide must then be cleaned up in additional purification steps. Protecting groups which can be eliminated while the oligonucleotide is still on the solid support, without the oligonucleotide being cleaved from the latter, are more advantageous. An example of such a protecting group is the para-nitrophenylethyloxycarbonyl protecting group, which can be removed with DBU while the oligonucleotide is still on the support. Subsequent cleavage of the oligonucleotide from the support with ammonia yields oligonucleotide which is already pure (F. Himmelsbach et al., Tetrahedron 40 (1984): 59).
A further criterion for the use of protecting groups for the exocyclic amino functions of the nucleobases is the stability toward acid conditions employed, as a rule, in each reaction cycle for eliminating the 5'-hydroxyl protecting group, for example, 2% dichloroacetic acid in dichloromethane. These conditions lead, particularly in the case of deoxyadenosine, to a not insubstantial degree of depurination. Cyclic diacyl groups, such as phthaloyl or succinoyl groups, were found, when used as protection for the exocyclic amino function, to be particularly stable toward depurination conditions (Kume et al., Tetrahedron Lett. 23 (1982): 4365; Nucleic Acids Res. 12 (1984): 8525; Nucleic Acids Res. Symp. Ser. 11 (1982): 26; Chemistry Letters (1983): 1597). However, these groups were also deprotected with ammonia (Kume et al., see above). An example of another cyclic diacyl group is the naphthaloyl group (Dikshit et al., Can. J. Chem. 66 (1988): 2989), which is likewise stable toward depurination and was also removed with ammonia.